Cancer cells are characterized by loss of contact inhibition and uncontrolled cell growth. Such modifications are triggered spontaneously or by noxae, co-called cancerogenes, which damage the genetic make-up. Such noxae include many chemicals, tobacco smoke, but also UV light. Besides that, genetic factors play a prominent role in the formation of cancer. Characteristic for cancer cells, beside their uninhibited growth, is also the tendency to form metastases in other organs.
It is of exceedingly high medical relevance to define prognosis factors for the progression of cancers, which provide information about the response to certain forms of treatment or are generally predictive for the occurrence of metastases, tumor progression and survival. So far, prognosis factors generally known to the person skilled in the art are used in medicine. These include, for example, the size of the tumor, its penetration depth into the surrounding tissue, cross-organ growth, the penetration into blood or lymphatic vessels or into lymph nodes, as well as the degree of differentiation of the tumor cells. In addition, some relatively unspecific serological markers exist.
The cell cycle of eukaryotic cells is generally subdivided into four phases: the G1-phase, in which the preparation for replication takes place, the S-phase, in which the DNA is synthesized and the actual cell functions take place, the G2-phase, in which the preparation for mitosis takes place, and the M-phase, the mitosis (FIG. 1). In addition, differentiated cells, which no longer divide, are described as being in the G0-phase. This organizational principle though is functional, but on closer inspection it becomes clear that the cell cycle is far more complex. Numerous processes must be initiated and activated, individual components joined and various cascades coordinated. For this reason, diverse control mechanisms exist, which ensure that any processes within the cell cycle are completed correctly. These control mechanisms are designated as “checkpoints1”. These are not fixedly defined points, as the word itself implies, but a reaction cascade, which can be initiated under certain circumstances. 1 Original definition (according to Weinert et al., The RAD9 gene controls the cell cycle response to DNA damage in Saccharomyces cerevisiae. 1988, Science 241:317-22): If a process B depends on the completion of a process A, then this dependency is conditional on a checkpoint, unless a mutation can eliminate the dependent relationship.
So far, several cell cycle checkpoints were characterized. The best investigated checkpoints in mammals are shown in FIG. 1. On the one hand, there is the DNA damage checkpoint, which can be activated by a damage of the DNA in different cell cycle phases. This damage can be caused by exogenous causes, like radiation, as well as by endogenous processes, e.g. spontaneous mutations. On the other hand, the replication checkpoint is activated by an incomplete or defective replication of the DNA. The spindle checkpoint monitors the formation of the bipolar spindle, the attachment of the kinetochores and the new formation of the centromere structures.
As long as these processes are not entirely completed or the damage eliminated, the entrance of the cell into the next cell cycle phase is inhibited to ensure that the genomic integrity of the cell is maintained (Elledge, S. J., Cell cycle checkpoints: preventing an identity crisis. 1996, Science 274:1664-72).
The most important task of a cell is to maintain genomic identity. Checkpoint kinase 1 is involved in essential control mechanisms in the cell cycle, which ensure that the transfer of defects to the daughter cell is minimized. The significant CHK1 reaction cascade at the G2/M checkpoint is shown in FIG. 2. The activation of CHK1 takes place due to DNA damages, which are mainly detected by the chromatin-bound Rad17 complex. Thereupon, the Rad17 complex recruits the Rad9-Hus1-Rad1 complex, which together with the ATR-Atrip complex activates CHK1, which is partially present in a chromatin-associated form, by phosphorylation. In that, ATR (Ataxia-telangiectasia- and Rad3-related) represents the most important activating component. It was shown that for the complete activation of CHK1, phosphorylation by the protein Claspin is also required. The activated CHK1 protein migrates from the cell nucleus into the cytoplasm, where in its turn it activates CDC25C (cell division cycle 25C) by phosphorylation. This process, on the other hand, enables the 14-3-3 protein (tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein) to bind to CHK1, so that it can return into the nucleus and remains there. In this manner, CDC2 (cell division controller 2) as well as the cyclin B complex are inhibited, which inhibits the entrance into mitosis. Subsequently, the DNA repair system can be initiated to eliminate the DNA damage (Jiang et al., Regulation of Chk1 includes chromatin association and 14-3-3 binding following phosphorylation on Ser345. 2003, J. Biol. Chem. 278:25207-17; Jeong et al., Phosphorylated claspin interacts with a phosphate-binding site in the kinase domain of Chk1 during ATR-mediated activation. 2003, J. Biol. Chem. 278:46782-8).
For CHK1, involvement in a checkpoint in the S-phase could also be verified. Here, upon defective replication, CHK1 is activated by ATM (Ataxia-telangiectasia mutated) by phosphorylation. For this checkpoint, too, additional activation by Claspin is required. The completely activated CHK1 now activates DNA protein kinases, together with which they phosphorylate p53 and thus can increase its activity. CHK1 is likewise able to phosphorylate TLK1 (tousled like kinase 1). This protein plays a decisive role in chromatin condensation, which, however, is inhibited by CHK1 to prevent progression in the cell cycle. Furthermore, CHK1 phosphorylates CDC25A (cell division cycle 25A) and thus initiates its degradation. As a consequence, the CDC protein is no longer able to activate the Cyclin complexes, due to which neither the S-phase can be advanced nor the M-phase started.